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| Status |
Public on Mar 17, 2017 |
| Title |
F0_Addict_2 |
| Sample type |
SRA |
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| Source name |
sperm
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| Organism |
Rattus norvegicus |
| Characteristics |
strain: SD
generation: F0
phenotype: Addict
cocaine exposure: Y
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Testes and cauda epidymides from mature male SDs were dissected, washed with PBS and placed in 35mm petri dishes containing HBSS supplemented with 20 mM HEPES pH 7.2, 1.2 mM MgSO4•7H2O, 1.3 mM CaCl2•2H2O, 6.6 mM sodium pyruvate, 0.05% lactate, 2 mM GlutaMAXT Supplement (Gibco®) and 0.5% BSA. Released sperm-containing supernatant was moved to 5 ml collection tubes and let stand for 10 min at 37℃ for swimming up. Supernatant was further filtered through 40 μm nylon mesh and laid on top of a three-layer gradient of 80%, 60% and 30% Percol, centrifuged for 10 min at 1,000 g at room temperature. Sperms were collected from 60% layer of the gradient, washed with the incubation buffer and cryopreserved. DNA was extracted from purified sperm by lysis with ,phenol-chloroform extraction followed by isopropanol precipitation and then preserved in TE buffer.
For library preparation, 1 mg genomic DNA was digested by MspI to generate short fragments that contain CpG dinucleotides at the ends. Then digested DNA was end-repaired, A-tailed and ligated to methylated Illumina adapters following the user manual of TruseqTM DNA Sample Prep Kit (Illumina, USA) Then, the CpG-rich DNA fragments (160-340 bp) are size selected, subjected to bisulfite conversion by MethylCode™ Bisulfite Conversion Kit (Life Technologies, USA) , and PCR amplified by ZymoTaqTM PreMix (Zymo, USA). Barcodes are indicated in the file names of each raw .fastq.gz file.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina Casava1.8 software used for basecalling.
The raw sequencing reads were quality checked and trimmed by Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) for low quality (Phred score < 30) nucleotides, adapters and artificially introduced bases in the end-repair step. Reads too short (< 20bp) were filtered.
Alignment to rat genome (RGSC Rnor_5.0/rn5) and methylation calling of the filtered reads were performed with Bismark. Parameters used were default except that 3 maximum mismatches in the "seed" were allowed and “paired-end” was specified. Bowtie short read aligner was used.
The bisulfite conversion rate was estimated by calculating the C to T conversion at non-CpG sites.
Differential methylation was calculated with BiSeq.
Genome_build: rn5
Supplementary_files_format_and_content: Bed files contains: the chromosome name, cytosine coordinate, cytosine coordinate (again), methylation value in percent, number of methylated reads (#M) and the number of unmethylated reads (#U). The file does not contain a header line. Coordinates are 1-based. Strand information does not need to be provided, but is inferred from the coordinates: Coordinates on the negative strand specify the cytosine base position (G on the positive strand). Coordinates referring to cytosines not in CpG content are automatically discarded.
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| Submission date |
Aug 26, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Qiumin Le |
| E-mail(s) |
qle09@fudan.edu.cn
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| Organization name |
Fudan University
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| Street address |
130 Dong'an Road
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| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE72401 |
Motivated cocaine seeking lead to transgenerational epigenetic inheritance of vulnerability to cocaine addiction |
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| Relations |
| BioSample |
SAMN04012630 |
| SRA |
SRX1164931 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1861858_High_F0_2.bed.txt.gz |
8.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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